beta -(THEOPHYLLINE ETHYL) MONOSULFITE

ABSTRACT

As a new compound Beta -(theophylline ethyl) monosulfite having the formula   AND SALTS THEREOF WITH INORGANIC AND ORGANIC BASES, THEIR PROCESS OF PREPARATION AND AS AN ANTIBACTERIAL AGENT, THE TETRACYCLINE SALT OF Beta -(THEOPHYLLINE ETHYL) MONOSULFITE.

United States Patent Clmence et al.

B-(THEOPHYLLINE ETHYL) MONOSULFITE Inventors: Francois Clmence, Rosny-sous- Boi; Odile Le Martret, Paris, both of France Assignee: Roussel Uclat', Paris, France Filed: Aug. 20, 1970 Appl. No.: 65,659

Related US. Application Data Continuation-impart of Ser. No. 730,628, May 20, 1968, abandoned.

Foreign Application Priority Data May 23, 1 967 I France "107454 July 28, 1967 France ..ll6l30 U.S. c1. ..'..260/253, 260/210 AB, 260 210 12, 260 210 K, 260/210 NE, 260 210 s, 260/242, 260/256, 260/559 AT, 424/1 80,

424/227, 424/253 Int. Cl. ..C07d 57/48, C07d 57/52 Field of Search ..260/210 AB,

210 5.210 K,2l0 NE Primary Examiner Alex Mazel Assistant Examiner-Anne Marie T. Tighe Attorney-Hammond & Littell [57] ABSTRACT As a new compound B-(theophylline monosulfite having the formula ethyl) l cu O=\N N/ and salts thereof 'with inorganic and organic bases their process of preparation and as an antibacterial agent, the tetracycline salt of B-(theophylline ethyl) monosulfite.

3 Claims, No Drawings B-(THEOPI-IYLLINE ETHYL) MONOSULFITE PRIOR APPLICATIONS The present application is a continuation-in-part application of our copending, commonly assigned U. S. Pat. application Ser. No. 730,628, filed May 20, 1968, now abandoned.

OBJECTS OF THE INVENTION novel method of treating microbial infections in warmblooded animals.

These and other objects and advantages of the invention will become obvious from the following description.

THE INVENTION The novel product of the invention is B-(theophylline ethyl) monosulfite which is a strong acid and which forms stable, well-defined salts with inorganic and organic bases and amino acids. The said acid and its salts are very soluble in water.

The salts of B-(theophylline I ethyl) monosulfite theophylline are useful in the paper industry and textile industry for impregnation or induction of cellulose fibers on articles to prevent proliferation of fungi, development of molds or bacteria and the occurrence of disagreeable odors in fermentation.

The said salts of the invention are also useful in the pharmaceutical industry since their excellent solubility in water permits the preparation of aqueous injectable solutions or of aerosols. Due to this water solubility, the injectable solutions have the advantage of greatly improved, activity, diffusibility and/or speed of action, particularly if it is a salt of a basic antibiotic. The said salts are also useful for preparing medicamental dressings of cellulose or artificial fibers when an external therapeutic action is desired.

Examples of suitable salts of B-(theophylline ethyl)- monosulfite are the alkali metal, alkaline earth metal, magnesium and aluminum salts and other therapeutically compatible metals; salts of aliphatic, arylaliphatic, cycloalkyl and heterocyclic amines, salts of basic antibiotics such as neomycin B, neomycin C, tetracycline, streptomycin, erythromycin, kanamycin, colimycin, hygromycin, hydroxymycin, etc.; salts of amino acids such as lysin, valine, phenylalanine, tyrosine, tryptophane, leucine, etc.; and salts of quaternary ammonium compounds such as choline tubocurarine, betaine, a-trimethylamino-e-amino-caproic acid, a,2- bis(trimethylamino)-caproic acid.

Examples of specific salts which are useful are sodium salt of fl-(theophylline ethyl) monosulfite, adenosin salt of B-( theophylline ethyl) monosulfite pyridoxine salt of B-(theophylline ethyl) monosulfite Z-dimethylamino-ethanol salt of B-(theophylline ethyl) monosulfite Z-diethyIamino-ethanol salt of B-(theophylline ethyl) monosulfite B-dimethylaminoethylbenzhydryl ether salt of B- (theophylline ethyl) monosulfite tetracycline salt of B-(theophylline ethyl) monosulfite lysin salt of B-(theophylline ethyl) monosulfite valine salt of B-(theophylline ethyl) monosulfite threonine salt of B-(theophylline ethyl) monosulfite betaine salt of B-(theophylline ethyl) monosulfite, and papaverine salt of B-(theophylline ethyl) monosulfite.

The novel process of the invention for the preparation of B-(theophylline ethyl) monosulfite comprises reacting 7-[B-chloroethyl]-theophylline with a metal sulfite to form the metal salt of B-(theophylline ethyl) monosulfite which is acidified to obtain the free acid.

The salts of B-(theophylline ethyl) monosulfite theophylline are prepared according to the usual methods of salification by using either a mineral or organic base or a salt thereof, the anion of which is easily isolable or eliminated, such as for example, a carbonate or a silicate. The salifrcation is preferably effected in water, but may be effected in an organic solvent in the presence or in the absence of water.

A preferred mode of the process of the invention comprises reacting 7-[B-chloroethyl]-theophylline with an alkali metal sulfite ata temperature greater than 60 C. The formation of the alkali metal salts is preferably effected by reaction of B-(theophylline ethyl) monosulfite with an alkali metal hydroxide, carbonate or bicarbonate. The formation of an organic base salt is preferably effected with a base such as 2- dimethylamino-ethanol, Z-diethyIamino-ethanol, or B- dimethylamino-ethylbenzhydryl ether. The formation of a heterocyclic base salt is preferably effected with a nitrogenated compound such as pyridoxine or adenosin. The formation of a basic antibiotic salt is preferably effected with an antibiotic such as strepto-. mycin or tetracycline. The formation of a quaternary ammonium salt is preferably effected with a quaternary ammonium hydroxide such as choline or a betaine such as betaine, a-trimethyl-amino -eaminocaproic acid, or a,2-bis-(trimethylamino)-caproic acid.

The novel antibiotic and antibacterial compositions of the invention are comprised of the tetracycline salt of B-(theophylline ethyl) monosulfite and a major amount of a pharmaceutical carrier. The compositions may be in the form of injectable solutions dispensed in ampoules, in multi-dose phials or as sterile powder for the extemporaneous preparation of injectable solutions, as tablets, coated tablets, aromatized powder, granules, emulsions, syrups, suppositories, ovules, intravaginal tablets, ointments, creams, nose and ear drops, eye-wash, mouth-wash, and topical pulverized powders. The said compositions are useful for the treatment of staphylococci, streptococci and infections caused by Gram-negative bacteria such as typhoid bacilli.

The method of combatting bacteria infections in warm-blooded animals comprises administering to warm-blooded animals having a bacteria infection an effective amount of tetracycline salt of B-(theophylline ethyl) monosulfite. The said product may be administered orally, transcutaneously, rectally or topically on the skin or mucosa. The usual useful dose is 3.5 to l3.5 mg/kg depending upon the method of administration.

ln the following examples there are described several preferred embodiments to illustrate the invention. However, it should be understood that the invention is not intended to be limited to the specific embodiments.

EXAMPLEl Preparation of B-(theophylline ethyl) monosulfite A solution of 72.6 gm of 7-[B-chlorethyl]-theophylline (obtained by the process described by DiPaco, Ann. Chim. Roma, Vol. 47, p'p;698-704, 1957) in 1,000 cc of water and a solution of 41.6 gm of sodium sulfite in 200 cc of water was refluxed for 7 hours and the resulting solution was filtered and evaporated to dryness under reduced pressure. The residue was taken up in 200 cc of hydrochloric acid and after filtering off the mineral precipitate, the filtrate was evaporated to dryness. The residue was washed with alcohol and dried to obtain 68 gm of B-(theophylline ethyl) monosulfite which was purified by dissolving in water and precipitating with acetone. The 65 percent yield of B-[theophylline ethyl] monosulfite has a melting point of 320 C. It occurred in the form of colorless crystals, very solublein water and insoluble in alcohol, acetone, ether and benzene. AnalysiszC H, N,O S; Calculated: C 37.50 percent 19.44 percent Found: 37.3 4.3 Infrared Spectrum (Nujol) Absence of NH at about 3,300 3,500 cm-l Presence of C=O at 1,675 cm Presence of RSO at 1,150 and 1,060 cm-l As far as is known, this compound is not described in the literature.

Molecular'weight 288.29

H 4.19 percent N EXAMPLE 11 Sodium salt'of B-(theophylline ethyl) monosulfite 28.8 gm of B-(theophylline ethyl) monosulfite dissolved in 65 cc of water were added to a solution of '4 gm of sodium hydroxide in cc of water to obtain an -alkaline pH of the order of 8. The solution was evaporated'to dryness under reduced pressure and the residue was washed with alcohol and dried to obtain 27.5 gm (Yield: 89 percent) of sodium salt of B- (theophylline ethyl) monosulfite in the form of colorless crystals, melting at about 340 C. Analysis: C,H N 0,NaS; molecular weight 310.28 Calculated: N .1 8.06 percent Found: 18.0

As far as is known, this compound is not described in the literature.

EXAMPLE lll Adenosin Salt of fi-(theophylline ethyl) monosulfite 2.67 gm of adenosin were added to a solution of 2.88 gm of B-(theophylline ethyl) monosulfite in cc of water, and after the solution was evaporated to dryness under reduced pressure, the residue was washed with alcohol and dried. The product thus obtained was purified by dissolving it in water and precipitating with acetone to obtain 3.14 gm (56 percent yield) of adenosin salt of B-(theophylline ethyl) monosulfite having a melting point of 178 C. The product occurred in the form of a microcrystalline compound, very soluble'in water and insoluble in alcohol and the usual organic solvents.

Analysis: C ,,H, N,O S; molecular weight 555.52 Calculated N 22.69 percent Found: 21.4

As far as is known, this compound is not described in the literature.

EXAMPLE IV Pyridoxine salt of B-(theophylline ethyl) monosulfite Using the procedure of Example 111, 2.88 gm of B- (theophylline ethyl) monosulfite and 1.69 gm of pyridoxine produced 4.3 gm (yield 72 percent) of crystals of pyridoxine salt of B-(theophylline ethyl) monosulfite which were purified by recrystallization from alcohol.

The product occurred in the form of colorless crystals, soluble in water and had a melting point of 169 C. Analysis: C H N O S molecular weight= 457.46 Calculated: N 15.31 percent Found: 15.3

As far as is known, this compound is not described in the literature.

EXAMPLE V Dimethylamino-ethanol salt of fi-(theophylline ethyl) monosulfite 1.78 gm of dimethylamino-ethanol were added to a solution of 5.76 gm of B-(theophylline ethyl) monosulfite in 12 cc of water and the solution was evaporated to dryness under reduced pressure. The residue was washed with isopropanol, dried and recrystallized from alcohol to obtain 5.4 .gm of dimethylamino-ethanol salt of B-(theophylline ethyl)monosulfite melting at 188 C. The product occurred in the form of colorless crystals soluble in water and insoluble in acetone. Analysis: C H N O S; molecular weight 377.42 Calculated: N 18.55 percent Found: 18.54

As far as is known, this compound is not described in the literature.

EXAMPLE Vl Diethylaminoethanol Salt of B-(theophylline ethyl) monosulfite Using the procedure of Example V, 2.88 gm of B- (theophylline ethyl)monosulfite and 1.17 gm of diethylamino-ethanol were reacted to obtain after recrystallization fromisopropanol 3.1 gmv (yield: 76.5 percent) of diethylamino-ethanol salt of B-(theophylline ethyl) monosulfite melting at 153 C.

The product occurred in the form of colorless crystals soluble in water and methanol and insoluble in acetone and ether. Analysis: C l-l,-,N O,S; Mo1ecularweight= 405.47 Calculated: N 17.27 percent Found: 17.36

As far as is known, this compound is not described in the literature.

EXAMPLE Vll B-dimethyl-amino-ethylbenzhydryl-ether salt of B- (theophylline ethyl) monosulfite Using the procedure of Example V, 2.88 gm of B- (theophylline ethyl) monosulfite and 2.55 gm of B- dimethyl-amino-ethylbenzhydryl-ether were reacted to obtain after recrystallization from alcohol, 3.68 gm (yield: 68 percent) of dimethyl-amino-ethylbenzhydryl-ether salt of B-(theophylline ethyl) monosulfite melting at 192 C.

The product occurred in the form of colorless crystals soluble in water. Analysis: C H N O S; molecular weight 543.62 Calculated: N 12.88 percent Found: 12.85 percent As far as is known, this compound is not described in the literature.

EXAMPLE Vlll dl-threonine salt of B-( theophylline ethyl)monosulfite 2.88 gm of B-(theophylline ethyl) monosulfite were dissolved in 9 cc of water and a solution of 1.19 gm of threo-a-amino-B-hydroxybutyric acid in 10 cc of water was added thereto. The solution was evaporated to dryness under reduced pressure and the residue was washed with alcohol and recrystallized from methanol to obtain 1.52 gm (Yield: 37 percent) of dl-th'reonine salt of B-(theophylline ethyl) monosulfite melting at 226 C.

The product occurred in the form of colorless crystals soluble in water. Analysis: C H N O S; molecular weight= 407.40 Calculated: N 17.19 percent Found: 17.08

As far as is known, this compound is not described in the literature.

EXAMPLE IX Lysin salt of B-(theophylline ethyl) monosulfite Using the procedure of Example Vlll, 2.88 gm of B- (theophylline ethyl) monosulfite and 1.46 gm of L(+)- lysin were reacted to obtain, after recrystallization from alcohol, 2.35 gm (yield: 56 percent) of lysin salt of B-(theophylline ethyl) monosulfite in the form of colorless needles very soluble in water and melting at 267 C. Analysis: C, H N O S: Molecular weight=437.47 Calculated: N 19.34 percent Found: 19.4

As far as is known, this compound is not described in the literature.

EXAMPLE X Valine Salt of B-(theophylline ethyl) monosulfite Using the procedure of Example V111, 2.88 gm of B- (theophylline ethyl) monosulfite and 1.17 gm of divaline were reacted to obtain, after recrystallization from alcohol, 2.39 gm (yield: 59 percent) of valine salt of B-(theophylline ethyl) monosulfite in the form of needles very soluble in water and melting at 202 C. Analysis: C l-l ',N O-,S: molecular weight 405.43 Calculated: N 17.27 percent Found: 17.0

As far as is known, this compound is not described in the literature.

EXAMPLE Xl Betaine Salt of B-( theophylline ethyl)-monosulfite Using the procedure of Example Vlll, 2.88 gm of B- (theophylline ethyl)-monosulfite and 1.17 gm of betaine were reacted to obtain, after recrystallization from alcohol, 2.63 gm (yield: 65 percent) of betaine salt of B-(theophylline ethyl)-monosulfite in the form of colorless crystals very soluble in water, slightly soluble in alcohol and melting at 253 C.

Analysis: C l-l N O S; Molecular weight 405 .43 Calculated: N 17.27 percent Found: 17.42

As far as is known, this compound is not described in the literature.

EXAMPLE X11 Calcium salt of B-(theophylline ethyl) monosulfite 5.76 gm of B-(theophylline ethyl) monosulfite were dissolved in 18 cc of water and 0.74 gm of calcium hydroxide were added thereto. After agitation, the dissolution was total and the solution was evaporated to dryness. The residue was taken up in the minimum of hot percent alcohol and after cooling, calcium salt of B-(theophylline ethyl) monosulfite crystallized.

. The product was separated, suction filtered and dried to obtain a yield of 86 percent pure product.

The said calcium salt occurred in the form of colorless crystals which were soluble in water and melted at a temperature greater than 380 C.

Analysis: C H CaN O S- molecular weight 614.66

Calculated: N 18.23 percent Found: 18.21 6.52

As far as is known, this compound is not described in the literature.

Ca 6.52 percent EXAMPLE x111 Magnesium salt of B-(theophylline ethyl) monosulfite Using the procedure of Example Xll, 2.88 gm of B- (theophylline ethyl) monosulfite and 0.29 gm of magnesium hydroxide were reacted to obtain, after crystallization from percent alcohol, an 81 percent yield of the magnesium salt of [El-(theophylline ethyl) monosulfite in the form of colorless crystals which were soluble in water and had a melting point greater than 360 C. Analysis: C l-l MgN O s molecular weight 598.87 Calculated: N 18.7 percent Mg 4.06 percent Found: 18.92 4.18

As far as is known, this compound is not described in the literature.

EXAMPLE XIV Papaverine Salt of B-(theophylline ethyl) monosulfite 2.88 gm of B-(theophylline ethyl) monosulfite were dissolved in 15 cc of water and under agitation 3.39 gm of papaverine were added thereto. The product dissolved rapidly and the solution thus obtained was evaporated to dryness. The residue was washed with alcohol and then recrystallized invthe hot and cold in 95 percent alcohol to obtain papaverine salt of B- (theophylline ethyl) monosulfite.

The product occurred in the form of yellow crystals soluble in water and melting at 237 C. Analysis: C H N O S; molecular weight 627.66 Calculated N l 1.16 percent Found: 11.22

As far as is known, this compound is not described in the literature.

EXAMPLE XV Tetracycline'salt of B-(theophylline ethyl) monosulfite 26.31 gm of tetracycline base were dissolved in 1,050 cc of methanol and 16.72 gm of B-(theophylline ethyl) monosulfite were added thereto. The solution was evaporated to dryness under reduced pressure and the residue was washed with ether and dried to obtain 43 gm of tetracycline salt of B-(theophylline ethyl) monosulfite. The product occurred in the form of a light yellow microcystalline powder soluble in water and methanol slightly soluble in ethanol, insoluble in ether, melting at about 250 C with decomposition and having a specific rotation [01],, l68 1 1 (C=l percent in water. Analysis: C H N O S; molecular weight 732.71 Calculated: N 1 1.47 percent Found: 11.49

As far as is known, this compound is not described in the literature.

PHARMACOLOGICAL STUDY TABLE I Tetracycline salt of B-(theophyllinc ethyl) Tetracycline monosulfite hydrochloride Administra- Dose Therapeutic Dose Therapeutic tion method in mg Action in mg Action Orally 1 100 0.66 100 Subcutaneously l 100 0.66 100 0.50 100 0.33 I

B. Typhus Bacilli Infection. 7

' Groups of 10 mice were infected experimentally by inoculation of a typhus bacilli culture and the animals then'received orally a single dose of tetracycline hydrochloride or tetracycline salt of B-(theophylline ethyl) monosulfite. The results reported in Table 11 show that the compound of the invention is at least equal to that of tetracycline hydrochloride.

TABLE 11 Tetracycline Salt of Tctrac cline fl-(theophylline ethyl) hydroch oride monosulfite Dose 5 Therapeu- Dose Therapeutic in mg tic action in mg action C. Hemolytic infections of Staphylococcus aureus Three groups of 10 Swiss strain mice weighing about 24 gm each were used in this experiment. The first lot served as controls and received intravenously 0.2 cc of an 18 hours culture of Staphylococcus aureus LW strain in Oxoid No. 11 medium. The second and third groups received intravenously 0.2cc of the same cul ture 20 minutes after the oral administration of tetracycline hydrochloride equal to 1 mg of tetracycline and the tetracycline salt of B-(theophylline ethyl) monosulfite equal to 1 mg of tetracycline respectively. The treatment was continued orally for 10 days at the rate of one intubation per day at the same dose.

On the 11th day, the animals were killed and the renal lesions in all mice were counted. The results are summarized in the following table.

TABLE III No. of mice No. of mice dead with visible Groups by l lth day renal lesions Controls 8 out of 10 5 Tetracycline hydrochloride 2 out of 10 i 5 Tetracycline salt of B-(theophylline ethyl) monosulfite 0 out of 10 2 The results of the above Table demonstrate the good tolerance and efficiency of the salt of the invention as compared to tetracycline hydrochloride.

Moreover, fewer visible renal lesions were found in the animals treated with the novel salt of the invention as compared to tetracycline hydrochloride.

D. Acute Toxicity The acute toxicity was determined on Swiss strain mice having an average weight of 18 to 22 gm by intravenously administering to groups of 10 mice increasing doses of the tetracycline salt of B-(theophylline ethyl) monosulfite and the LD was calculated by the method of Dragstaedtand Lang to be 296 mg/kg.

E. Sub-acute toxicity Rats of about 140 gm received daily by an esophogus tube for 21 days increasing doses of the said tetracycline salt in 1 cc of water and control rats received only the 1 cc of water. The animals weight was measured every day and at the end hematological tests were conducted on groups of three rats per dose. The results in the next tables show that the said product does not affect the ponderal growth nor disturb the hemopoietic and leuco-forming organs.

TABLE IV Control Normal 480 mgJkg. 2-10 ing/kg. U6 mgJkg. tests formula Monocytcs, percent 1. 6 0. 5 0. 7 2. 3 0-2 lolyuuclem' ncutrophilcs, pcrcc11t 20 31. 9 21. 7 33. 6 30-35 lolynuclear cosinophilcs, pvrccnL 1.11 0.5 0.7 1.1 0-2 Polynuclcur basophilcs, percent. U 0 0 0 0-4 'lAHLIC V Controls 48 nigJkt'. 240 mg./kg. 1h mgjkg Number Average Number Average Number Average Ninnhei Average of rats weight, g. of rats weight, g. 01 rats weight, g. of rats weight, g.

Initial Weight 1 13:) 111 147 10 141 11) 134 1st ingestion. 10 137 10 14'.) 10 143 10 138 2nd ingestion 10 153 10 158 10 155 10 147 3rd ingestion 10 169 10 17.) 10 171) 11) 165 4th ingestion 10 166 10 176 10 179 10 163 5th ingestion 176 10 1825 10 187 10 173 6th ingestion 10 174 10 186 10 195 10 180 7th ingestion 10 187 10 194 10 204 10 186 8th ingestion 1 201 10 213 10 .220 10 201 11th ingestion 1 204 11) 313 10 1.11) 10 200 10th ingestion 1 211 111 211) 1(1 2211 10 213 111.11 ingestion J 215 111 223 10 .2211 10 211 121.11 ingestion J 211) 11) 3 10 .237 10 22% 1311.1! ingestion J 222 11] .3111 10 244 10 231 141.11 ingesflon t, '1 .107 11] 1-11 11) 247 10 234 15th ingestion I 237 11) 24H 10 251 111 1.43 113th ingestion J 237 11) .2111 11) 255 111 212 17th ingestion. .1 241) 11) .252 11) .2511 10 2415 18th ingestion .1 254 10 .271 111 27G 11) .263 111111 ingostioiL .1 .257 11] 27 1 .1 21-11) 11) 268 2011i ingestion. J 261 111 .578 'J 282 10 261) 21st ingestion. .1 2113 111 .281 o .1 5 10 271 increase of Wei +114. 8 +11 .5 +1112. 2 +1112. 3

Various modifications of the compositions and method of the invention may be made without departing from the spirit or scope thereof and it is to be understood that the invention is intended to be limited only as defined in the appended claims.

We claim:

1. A compound selected from the group consisting of B-(theophylline ethyl) monosulfite having the formula CHz-CIHOS 02H L CH 0: N N 

1. A compound selected from the group consisting of Beta -(theophylline ethyl) monosulfite having the formula
 2. A compound of claim 1 which is Beta -(theophylline ethyl) monosulfite. 